Thioredoxin is a small protein present in all living cells. The diverse functions of thioredoxin include a role in DNA synthesis, carbohydrate metabolism and hormone action. The clinical relevance of thioredoxin is underscored by recent observations that the human gene encoding thioredoxin responds to virally induced transformation. HTLV-I transformed human T lymphocytes, human B lymphocytes immortalized by Epstein-Barr virus, and in addition chicken embryo fibroblasts transformed by Rous sarcoma virus, all show increased accumulation of thioredoxin transcripts. The relationship between thioredoxin and transformation is not understood. However one possible explanation is that thioredoxin represents a rate limiting step in the synthesis of deoxyribonucleotides and that rapidly dividing cells require elevated levels of thioredoxin to meet the needs of DNA replication. Thioredoxin may be one of perhaps several factors that supply the reducing power to ribonucleotide reductase. The goal of this application is to gain new insights into the importance of thioredoxin for cell growth and division in eukaryotes. Towards this goal the project combines biochemical and genetic approaches to understand the cellular response to thioredoxin deficiency in a mutant of the yeast Saccharomyces cerevisiae. This mutant lacks the two known thioredoxin genes of this organism. Deletion of the two thioredoxin genes perturbs the progression of the cell through the cell cycle. The period of DNA replication (S phase) is prolonged and the GI phase is shortened. The first aim of this project is to provide direct evidence that the delay in S phase is a result of a decreased rate of deoxyribonucleotide synthesis. Both the levels of ribonucleotide reductase activity and deoxyribonucleotides will be assayed. Ribonucleotide reductase RNR2 mRNA levels will also be analyzed. To test if the integrity of DNA replication is impaired the rate of spontaneous mutation and the rate of mitotic recombination will be measured. In addition, I will determine whether viability of the yeast mutant is dependent upon DNA repair pathways or a cell cycle checkpoint (RAD9 ) that responds to DNA damage. Finally, since the double thioredoxin mutant is viable, an alterative pathway for production of deoxyribonucleotides must exist in yeast. This pathway will be investigated by in vitro methods and mutant analysis.